A major problem with recombinant tissue plasminogen activator (rt-PA) for ischemic stroke is the limited time window for effective therapy and the related complications of cerebral hemorrhage and edema. In response to RFA-HL-05-004, we propose a series of translational animal experiments to test the idea that simultaneously inhibiting matrix metalloproteinases (MMPs) will reduce rt-PA complications, and significantly increase the time window for reperfusion therapy. In Aim 1, we will use a well characterized rat model of embolic (clot-based) focal cerebral ischemia to test the effects of combining rt-PA with the broad spectrum MMP inhibitors BB-94 and minocycline. We will assess the effects of different times of treatment 3-12 h post-ischemia to see whether MMP inhibition can lengthen the time window for effective rt-PA reperfusion. In Aim 2, we will assess the use of immunoliposomes to target our MMP inhibitors to areasof neurovascular damage. Because MMPsalso participate in normal physiology, this form of targeting may be required to optimize clinical use. We will test anti-actin and anti-adhesion molecule (e.g. ICAM-1, E-selectin) immunoliposomes that target cells with damaged membranes and activated ("inflammed") endothelium and astrocytes. In the rat model of embolic clot stroke, we will compare the effects of "naked" BB-94 and minocycline versus immunoliposome- encapsulated compounds. Finally, in Aim 3, we will use in vivo 2-photon imaging to assess rt-PA, MMP inhibition, and vascular leakage within cranial windows in the rat model of embolic stroke. DQ-FITC-gelatin will be infused into the cranial windows, then in vivo 2-photon imaging will be used to mapneurovascular MMP activity overtime. Blood-brain barrier integrity will be assessed using fluorescent dextrans of different sizes. We will compare MMP profiles after saline, rt-PA, or rt-PA plus MMP inhibitors. Over the past 6 years, our lab has accumulated cellular and mechanistic data implicating MMPs in rt-PA thrombolytic complications after stroke. We are now ready to perform the required translational studies derived from our basic findings. If we can obtain proof-of-concept in our animal models that rt-PA plus MMP inhibitors can significantly lengthen the time window for safe and effective reperfusion, this may be an important step forward for combination stroke therapy.